Cartilage matrix protein and methods for use

ABSTRACT

DNA constructs coding for a chimeric polypeptides containing fragments of cartilage matrix proteins that can bind collagen and their protein products are described. Also, the invention relates to purified chimeric polypeptides, and methods of their production and purification from transformed cells as well as their use as agents in therapeutics and clinical imaging. In addition, the invention disclosed a method for forming collagen fibrils using the chimeric polypeptide.

BACKGROUND OF THE INVENTION

This invention was made with Government support under Contract #ND 22016and DK 28433 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

This application is a continuation of application Ser. No. 08/006,096filed on Jan. 15, 1993, now abandoned which is a continuation in part ofapplication Ser. No. 07/866,403 filed Apr. 10, 1992, now abandonedEntitled: CARTILAGE MATRIX PROTEIN AND METHODS FOR USE.

This invention relates to cartilage matrix protein.

Cartilage matrix protein (CMP) is a noncollagenous protein of theextracellular matrix of cartilage. CMP is a homotrimer of disulfide bondlinked subunits.

Argraves et al. (Proc. Nat'l. Acad. Sci. USA 84:464, 1987) and Kiss etal. (J. Biol. Chem. 264:8126, 1989) disclose that chicken CMP includes adomain (residues 148-183) with significant homology to epidermal growthfactor and two homologous repeat sequences (residues 30-220 and262-450). The homologous repeats (CMP-1 and CMP-2 respectively) arehomologous to regions within von Willebrand factor, complement factor B,complement factor C2, type VI collagen, and the α chains of theintegrins Mac-1, p150,95 and LFA-1 (see Kiss et al., supra for a review).

Human CMP is 79% identical to chicken CMP overall; human and chickenCMP-1 domains are 79% identical; human and chicken CMP-2 domains are 84%identical (Jenkins et al., J. Biol. Chem. 265:19624, 1990).

CMP may interact with both proteoglycan and collagen (Goetinck et al.,Annals of the New York Academy of Sciences 599:29, 1990).

Bonaldo et al. (Biochemistry 29:1245, 1990) report that type VI collagenhas several repeats homologous to CMP and that a region which includesthese repeats is involved in interactions between type VI collagen andtype I collagen.

SUMMARY OF THE INVENTION

In general, the invention features a polypeptide (preferablysubstantially pure) which is a fragment or analog of cartilage matrixprotein, the polypeptide being capable of binding collagen.

In various preferred embodiments: the polypeptide includes CMP-1 orCMP-2; the polypeptide includes a fragment or analog of CMP-1 or CMP-2;the polypeptide is or comprises all or part of CMP-1, e.g., anN-terminal fragment of CMP-1 approximately 45 amino acids in length, orthe approximately 8-10 residue collagen binding sequence 1 (CBS1,defined in detail below) motif; the polypeptide is or comprises all orpart of CMP-2, e.g., a N-terminal fragment of CMP-2 approximately 45amino acids in length, or the approximately 8-10 residue CBS1 motif ofCMP-2; the polypeptide is or comprises a CBS1 motif; and the polypeptideis all or part of CMP-1 and CMP-2.

In a related aspect, the invention features a method for formingcollagen fibrils. The method includes contacting cartilage matrixprotein, or a polypeptide of the invention, with collagen.

In preferred embodiments the polypeptide is fragment or analog ofcartilage matrix protein; is a fragment or analog of cartilage matrixprotein domain CMP-1 or CMP-2; includes or consists essentially of aCBS1 motif; includes or consists essentially of an N-terminal fragmentof CMP-1 approximately 45 amino acids residues in length; consistsessentially of or includes the CBS1 motif of CMP-1; includes or consistsessentially of an N-terminal fragment of CMP-2 approximately 45 aminoacid residues in length; consists essentially of or includes the CBS1motif of CMP-2.

In another related aspect, the invention features a method fordelivering a compound to a tissue, e.g., a collagenous tissue, themethod includes administering to a patient the compound linked to apolypeptide of the invention.

In preferred embodiments the polypeptide is fragment or analog ofcartilage matrix protein; is a fragment or analog of cartilage matrixprotein domain CMP-1 or CMP-2; includes or consists essentially of aCBS1 motif; includes or consists essentially of an N-terminal fragmentof CMP-1 approximately 45 amino acids residues in length; consistsessentially of or includes the CBS1 motif of CMP-1; includes or consistsessentially of an N-terminal fragment of CMP-2 approximately 45 aminoacid residues in length; consists essentially of or includes the CBS1motif of CMP-2.

In another aspect, the invention features a molecular conjugate whichincludes a polypeptide which is collagen-binding fragment of cartilagematrix protein and an imaging or therapeutic agent.

In preferred embodiments the polypeptide is fragment or analog ofcartilage matrix protein; is a fragment or analog of cartilage matrixprotein domain CMP-1 or CMP-2; includes or consists essentially of aCBS1 motif; includes or consists essentially of an N-terminal fragmentof CMP-1 approximately 45 amino acids residues in length; consistsessentially of or includes the CBS1 motif of CMP-1; includes or consistsessentially of an N-terminal fragment of CMP-2 approximately 45 aminoacid residues in length; consists essentially of or includes the CBS1motif of CMP-2.

In another aspect the invention features a method of promoting theattachment of collagen to a surface including coating the surface with acollagen binding fragment of collagen matrix protein and contacting saidcoated surface with collagen.

In preferred embodiments the polypeptide is fragment or analog ofcartilage matrix protein; is a fragment or analog of cartilage matrixprotein domain CMP-1 or CMP-2; includes or consists essentially of aCBS1 motif; includes or consists essentially of an N-terminal fragmentof CMP-1 approximately 45 amino acids residues in length; consistsessentially of or includes the CBS1 motif of CMP-1; includes or consistsessentially of an N-terminal fragment of CMP-2 approximately 45 aminoacid residues in length; consists essentially of or includes the CBS1motif of CMP-2.

In preferred embodiments the surface is a surface of a device to beimplanted in the body of a recipient.

In another aspect the invention features a substantially purified DNAencoding a polypeptide of the invention; a vector including a DNAsequence of the invention; a cell containing DNA encoding a polypeptideof the inventions e.g., cell capable of expressing the polypeptide; anessentially homogeneous population of cells, each of which comprises theDNA encoding a polypeptide of the invention; and a polypeptide producedby expression of DNA encoding a polypeptide of the invention.

In another aspect the invention includes a therapeutic compositionincluding a polypeptide of the invention and a pharmaceuticallyacceptable carrier.

In another aspect the invention features a method for manufacture of apolypeptide of the invention including culturing a cell containing DNAencoding a polypeptide of the invention in medium to express thepolypeptide.

The nucleotide sequence of chicken CMP is available from GenBank™/EMBLData Bank under accession numbers X12346-X12354. The nucleotide sequenceof human CMP is available from GenBank™/EMBL Data Bank under accessionnumbers Jo5666 and JO5667.

CMP-1 (the CMP-1 domain) is a sequence corresponding to amino acids30-220 of chicken CMP; CMP-2 (the CMP-2 domain) is a sequencecorresponding to amino acids 262-450 of CMP (numbering according to Kisset al., supra). CMP-1 also corresponds to amino acids 23 to 222 of humanCMP, and CMP-2 also corresponds to amino acids 264-453 of human CMP(numbering according to Jenkins et al., supra). The term CMP-1 alsoincludes polypeptides corresponding to domains in proteins such as theyon Willebrand factor, complement factor B, complement factor C2, typeVI collagen, and the α chains of the integrins Mac-1, p150,95 and LFA-1that are homologous the to human or chicken CMP-1 domain. The term CMP-2also includes polypeptides corresponding to domains in proteins such asthe von Willebrand factor, complement factor B, complement factor C2,type VI collagen, and the α chains of the integrins Mac-1, p150,95 andLFA-1 that are homologous to the human or chicken CMP-2 domain. Suchhomologous domains have been identified by standard techniques (see Kisset al., supra for a review). Such domains are 70% homologous, preferably80%, more preferably 90% homologous to the human CMP-1 domain or thehuman CMP-2 domain.

The CBS1 motif is an 8-10 amino acid sequence found in CMP-1, CMP-2, anda large number of other proteins, some or many of which bind collagen.The CBS1 motif is located at amino acids 38-47 (in CMP-1) (TDLVFIIDSS)(SEQ. ID NO: 1) and 271-280 (in CMP-2) (LDLVFLIDGS) (SEQ. ID. NO. 2) ofchicken CMP (numbering according to Kiss et al., supra). The CBS1 motifcorresponds to amino acids 40-49 (in CMP-1) (TDLVFVVDSS) (SEQ. ID. NO:3) and 274-283 (in CMP-2) (TDLVFLIDGS) (SEQ. ID. NO: 4) of human CMP(numbering according to Jenkins et al., supra). The CBS1 motif alsoincludes the homologous 8-10 amino acid residue sequence found inCMP-1-like and CMP-2-like domains of other proteins, particularlycollagen binding proteins such as the von Willebrand factor, complementfactor B, complement factor C2, type VI collagen, and the α chains ofthe integrins Mac-1, p150,95 and LFA-1. Such homologous domains havebeen identified by standard techniques (see Kiss et al., supra for areview). Such domains are preferably 60-70% homologous, more preferably80%, most preferably 90% homologous to the human CBS1 of the CMP-1 orCMP-2 domain. The invention includes the CBS1 domains from the followingproteins (the protein is given first then residues 2-10 of the CBS1domain is listed): α1 (XII) vW1, DIVFLTDAS (SEQ ID NO: 5); α1 (XII) vW2,DIVLLVDGS (SEQ ID NO: 6); α1; α1 (XII) VA, DLVFLVDGS (SEQ ID NO: 7); α1(XII) VB, DWFLVDGS (SEQ ID NO: 8); α1 (XIV) vW1, DLVFLVDGS (SEQ ID NO:9); CMP1; CMP1, DLVFIIDSS (SEQ ID NO: 10); CMP2, DLVFLIDGS (SEQ ID NO:11); α1 (VI) A'1, DIMLLVDSS (SEQ ID NO: 12); α1 (VI) A'2, DLLFVLDSS (SEQID NO: 13); α1 (VI) A'3, DLFFVLDTS (SEQ ID NO: 14); α2 (VI) D3,DIVFLLDGS (SEQ ID NO: 15); α2 (VI) D2, DIMFVIDSS (SEQ ID NO: 16); α3(VI) A'1, DIAFIMDSS (SEQ ID NO: 17); α3 (VI) A'2, ELAFAIDTS (SEQ ID NO:18); α3 (VI) A'3, DVILGFDVS (SEQ ID NO: 19); α3 (VI) A1, DIVFLLDGS (SEQID NO: 20); α23; α3 (VI) A2, DWFLIDSS (SEQ ID NO: 21); α3 (VI) A3,DWFLVDGS (SEQ ID NO: 22); α3 (VI) A4, DWFLIDGS (SEQ ID NO: 23); α3 (VI)A5, DILFLIDGS (SEQ ID NO: 24); α3 (VI) A6, DIIFLLDGS (SEQ ID NO: 25); α3(VI) A7, DIVFLIDGS (SEQ ID NO: 26); α3 (VI) A8, DLIFLIDGS (SEQ ID NO:27); HUM vWF A1, DLVFLLDGS (SEQ ID NO: 28); HUM vWF A2, DVAFVLEGS (SEQID NO: 29); HUM vWF A3, DVILLLDGS (SEQ ID NO: 30); HUM Mac-1, DIVFLIDGS(SEQ ID NO: 31); HUM p150,95, DIVFLIDGS (SEQ ID NO: 32); HUM LFA1,DLVFLFDGS (SEQ ID NO: 33); RAT VLA-1, DIVIVLDGS (SEQ ID NO: 34); HUMVLA-2, DWLVCDES (SEQ ID NO: 35); HUM VLA-4, DISFLLDVS (SEQ ID NO: 36).

Fragments or analogs of CMP capable of binding collagen can beidentified using the assay described below.

Fragments or analogs of CMP useful for forming collagen fibrils includethose which have an effect on fibril formation as assayed by thefibrillogenesis assay described below. Any fragment or analog whichsubstantially affects fibrillogenesis will cause a change in the opticaldensity profile in this assay. Microscopic examination (described below)of the resulting fibrils can then be used to determine the exact effectof the CMP fragment or analog on fibril morphology.

The invention includes CMP and CMP polypeptides which are substantiallyhomologous to human CMP as well as other naturally occurring mammalianand avian CMP. Also included are: allelic variations; natural mutants;and induced mutants; Also included are CMP and CMP polypeptides encodedby DNA that hybridizes under high or low (e.g., washing at 2×SSC at 40°C. with a probe length of at least 40 nucleotides) stringency conditionsto a nucleic acid naturally occurring (for other definitions of high andlow stringency see Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1989, 6.3.1 -6.3.6, hereby incorporated by reference).

The invention also includes analogs of naturally occurring CMPpolypeptides. Analogs can differ from naturally occurring CMP by aminoacid sequence differences or by modifications that do not affectsequence, or by both. Analogs of the invention will generally exhibit atleast 70%, more preferably 80%, more preferably 90%, and most preferably95% or even 99%, homology with all or part of a naturally occurring CMPsequence (intact CMP, CMP-1 or CMP-2). The length of comparisonsequences will generally be at least about 6-8 amino acid residues,usually at least 20 amino acid residues, more usually at least 24 aminoacid residues, typically at least 28 amino acid residues, and preferablymore than 35 amino acid residues. Modifications include in vivo, or invitro chemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.,those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps,e.g., by exposing the polypeptide to enzymes that affect glycosylationderived from cells that normally provide such processing, e.g.,mammalian glycosylation enzymes. Also embraced are versions of the sameprimary amino acid sequence that have phosphorylated amino acidresidues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.Analogs can differ from naturally occurring CMP by alterations of theirprimary sequence. These include genetic variants, both natural andinduced. Induced mutants may be derived by various techniques, includingrandom mutagenesis of the encoding nucleic acids using irradiation orexposure to ethanemethylsulfate (EMS), or may incorporate changesproduced by site-specific mutagenesis or other techniques of molecularbiology. Also included are analogs that include residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring or synthetic amino acids, e.g., β or γ amino acids. Peptideswith N-terminal or C-terminal modifications to enhance peptide stabilityare also within the invention. Cyclic forms of the polypeptides of theinvention are within the invention.

In addition to substantially full-length polypeptides, the inventionalso includes biologically active e.g., collagen binding, fragments ofthe polypeptides. As used herein, the term "fragment", as applied to apolypeptide, will ordinarily be at least about 6-10 contiguous aminoacids, typically at least about 20 contiguous amino acids, moretypically at least about 30 contiguous amino acids, usually at leastabout 40 contiguous amino acids, preferably at least about 50 contiguousamino acids, and most preferably at least about 60 to 80 or morecontiguous amino acids in length. Fragments of CMP can be generated bymethods known to those skilled in the art. The ability of a candidatefragment to exhibit a biological activity of CMP can be assessed bymethods known to those skilled in the art. Also included are CMPpolypeptides containing amino acids that are normally removed duringprotein processing, including additional amino acids that are notrequired for the biological activity of the polypeptide, or includingadditional amino acids that result from alternative mRNA splicing oralternative protein processing events.

The invention also includes DNA, preferably substantially pure DNA,encoding the polypeptides of the invention. The invention also includesantibodies, e.g., monoclonal antibodies, directed against thepolypeptides of the invention. The invention also includes a chimericpolypeptide which includes a polypeptide of the invention.

A CMP polypeptide, fragment, or analog is biologically active if itexhibits a biological activity of a naturally occurring CMP, e.g.,binding collagen or affecting fibrillogenesis.

Substantially pure DNA is DNA that is not immediately contiguous withboth of the coding sequences with which it is immediately contiguous(i.e., one at the 5' end and one at the 3' end) in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived.

Homologous refers to the sequence similarity between two polypeptidemolecules or between two nucleic acid molecules. When a position in bothof the two compared sequences is occupied by the same base or amino acidmonomeric subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The homology between two sequences is a function of the number ofmatching or homologous positions shared by the two sequences. Forexample, 6 of 10, of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology.

A substantially pure preparation of a polypeptide is a preparation whichis substantially free of the proteins with which it naturally occurs ina cell.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

Brief Description of the Drawings

The drawings are first briefly described.

FIG. 1 is a graph which depicts the results of a fibrillogenesis assay.Optical density (x1000) at 400 nm is plotted as a function of time (min)for reactions carried out in the presence (open squares) and absence(filled diamonds) of cartilage matrix protein).

FIG. 2 is a graph which depicts the results of a collagen binding assay.Relative binding is plotted as a function of CMP concentration (μg/ml).

FIG. 3 depicts the amino acid sequence of human CMP. (SEQ ID NO: 37)

Cartilage Matrix Protein

Described below are methods for producing CMP in bacterial cells,generating CMP fragments, determining whether a given CMP fragment bindsto collagen, determining whether a given CMP fragment influencesfibrillogenesis, as well as methods for using CMP and fragments thereof.

Binding Studies Demonstrate the Interaction of CMP and Type II Collagen

An ELISA, performed as described below, demonstrated that CMP interactswith type II collagen. This assay was also used to demonstrate thebinding of reduced and alkylated cartilage matrix protein to type IIcollagen. CMP and reduced and alkylated CMP bound in aconcentration-dependent manner, as detected by an anti-cartilage matrixmAb (III-D5). The interaction between CMP and collagen could also bedemonstrated when CMP was immobilized and the binding of soluble type IIcollagen was detected with a mAb which recognizes type II collagen(I-B4). The binding of collagen to immobilized CMP was inhibited bysoluble CMP, demonstrating that the interaction is specific. Reduced andalkylated CMP was equally effective in this inhibition. Bovine serumalbumin did not inhibit binding in this assay, whereas relatively highconcentrations of link protein showed a minor inhibition which could bedue to residual traces of CMP in the link protein preparation. However,link protein has previously been shown to bind to types I and IIcollagen (Chandrasekhar et al., J. Biol. Chem. 258:6226, 1983) and, athigh levels, this binding may interfere with the binding of CMP tocollagen.

Bacterial CMP Expression

The isolation of partial cDNA clones of CMP has been previouslydescribed, and the entire CMP coding sequence was determined from thesecDNA clones and from sequencing of the exonic fragments of a genomicclone (Argraves et al., supra; Kiss et al., supra). The longest cDNAclone pCMP4 (1.304 nucleotides long) spanned an EcoRI site 53nucleotides 3' to the translation termination codon and extended in the5' direction to contain 1,251 out of 1,482 nucleotides of the codingsequence. A full-length CMP cDNA was constructed by using anoligonucleotide primer overlapping a unique Sst I site near the 5' endof pCMP4 clone to synthesize a first strand cDNA extending from the SstI site to the 5' end of the mRNA. A second oligonucleotide whichoverlaps the AUG initiation codon of CMP and generates a restrictionsite to facilitate cloning of the CMP cDNA in the same reading frame asthe α-galactosidase gene of pUC119 was then used to amplify thisfragment by the polymerase chain reaction (PCR). This PCR product wascloned as a KpnI-SstI fragment into the same sites of pUC119 andsequenced to insure that no mutations were inadvertently introducedduring the reverse transcriptase or the PCR reactions. This clone isreferred to as pCM5P'. Subsequently, the SstI-EcoRI fragment of pCMP4was gel purified and cloned in the SstI-EcoRI site of pCMP5' to generatethe full length CMP cDNA clone, pCMP-F1. For expression of full lengthmature CMP protein, the fragment of pCMP-F1 from the mature end of theprotein (Alanine 24) to the end of the CMP cDNA insert was amplified byPCR. The 5' PCR oligomer included an ATG initiation codon incorporatedas part of an NcoI site; the 3' PCR oligomer also introduced an NcoIsite at the 3' end of the PCR fragment. This fragment was purified, cutwith NcoI and cloned into the NcoI site of the E. coli expression vectorpET-11d (Novagen). The clone pET-CMP 1 containing the insert in thecorrect orientation was selected and sequenced to insure thatinadvertent mutations were not introduced during the cloning procedure.The E. coli strain BL21(D3)pLysS (Novagen) was transformed with pET-CMP1 and at appropriate times CMP expression was induced byisopropyl-β-D-thiogalactopyranoside.

Antibodies

Polyclonal antisera were generated in rabbits against CMP and asynthetic peptide corresponding to residues Phe³⁸⁰ -Val⁴²⁴ of CMP (thenumbers identifying the amino acids refer to the residue numbers in theprimary translation product) conjugated to keyhole limpet hemocyanin, asdescribed by Goetinck et al. (J. Cell Biol. 105:2403, 1987). Monoclonalantibodies were raised against a crude guanidine hydrochloride extractof sternal cartilage from 4-6 week-old chickens. ELISA screening showedthat mAb III-D5 recognized CMP and I-B4 recognized type II collagen.

For use in electron microscopy, III-D5 ascites fluid and rabbitanti-Phe³⁸⁰ -Val⁴²⁴ serum were purified by affinity chromatography on acolumn of CMP-Sepharose. Polyclonal antisera to chicken cartilage typeII collagen and aggrecan used in immunofluorescence studies were asdescribed previously (Vertel et al., Natl. Acad. Sci USA 76:1261, 1979;Upholt et al., Proc. Nat'l. Acad. Sci, USA 76:4847, 1979) .

Binding Assays

The interaction of CMP with collagen was studied using ELISA.Immobilization of CMP to microtitration plates (EIA, Linbro; FlowLaboratories, McLean, Va.) was carried out in 0.05M sodium carbonatebuffer, pH 9.6 (60 μl/well), overnight at room temperature. Collagens(Type I, Collaborative Research Inc., Bedford, Mass.; Type II NittaGelatin Inc., Osaka, Japan; Types III, IV, and V, Calbiochem Corp., LaJolla, Calif.; type VI, Telios Pharmaceuticals, San Diego, Calif.; C1q,Center for Blood Research, Boston, Mass.) were immobilized in the samebuffer by allowing the coating solution to dry at 37° C., overnight. Allsubsequent additions were for 1 hr in phosphate buffers saline,containing 0.05% Tween-20 (PBS-T). After each addition, plates werewashed with PBS-T. The binding of collagen to plastic or to immobilizedCMP was detected with mAb I-B4. The binding of CMP to plastic or toimmobilized collagen was detected with mAb III-D5. The detectingmonoclonals were followed in each case by peroxidase-conjugated rabbitanti-mouse IgG (H+L) and then peroxidase-substrate (BioRadLaboratories). Absorbance at 405 nm was determined and recorded using amicrotitration-plate reader (Titertek Multiskan Plus, Flow Labs Inc.,McLean, Va.).

Inhibition of collagen-CMP binding was undertaken by mixing variousconcentrations of inhibitor with collagen and allowing the mixture tostand for one hour prior to adding to CMP coated plates. Bound collagenwas detected as described above.

Rotary Shadowing Studies Reveal the Molecular Interaction of CMP andtype II Collagen

In order to characterize the molecular sites of interaction of CMP andcollagen, electron microscopic studies, using rotary shadowing of thecomponents (performed as described below), were initiated. Isolated CMPmolecules appeared as globular proteins comprised of two or threespherical domains connected by bent rods. Variations that were sometimesobserved may be due to different angles of view. Isolated type IIcollagen molecules showed a typical rod-like structure of 280 nm inlength. In mixtures of CMP and type II collagen, CMP was localized atboth ends of the collagen molecule, resulting in the formation ofconcatenates. In addition to simple concatenation, networks of collagenmolecules were also observed, with CMP-globules at the branch points.

Rotary Shadowing

Preparations of purified CMP or collagen were dialyzed against 0.1Mammonium acetate (HPLC grade, Sigma) pH 7.4, mixed with glycerol (40%)to a concentration of 25-50 μg/ml and nebulized onto freshly cleavedmica substrates (Engvall et al., J. Cell. Biol. 102:703, 1986). Eachsubstrate was attached to a rotary stage in an Edwards E 306 vacuumevaporator (pumped to 10⁻⁶ Torr) at an angle of 5-10° to the twinelectron beam source approximately 10 cm from the sample. Replicas weregenerated by evaporation of pure tungsten deposited to a thickness of19-30 Å, as measured by a water cooled (quartz crystal) film thicknessmonitor (FTM4) (Peters, B. Electronenmikroskop. Direktabh. Oberfl.12:377, 1979). The resulting films were backed by carbon evaporation,floated on a clean water surface, and picked up on 300 mesh grids forelectron microscopy. Samples were viewed and photographed in an HitachiH-600 STEM.

Nanomelia embryos were obtained from fertile eggs resulting from matingsbetween parents heterozygous for the nanomalic mutation. These eggs wereobtained from the Department of Animal Genetics, the University ofConnecticut, through the courtesy of Dr. Louis J. Perro.

Immunolocalization Studies Demonstrate the Co-Localization of CMP andType II Collagen in the Cartilage Extracellular Matrix

The co-localization of CMP with type II collagen and aggrecan wasexamined in 5-day-old cultures of chicken sternal chondrocytes usingdouble immunofluorescent staining reactions as described below.Chondrocytes were grown in the presence of ascorbic acid to facilitatethe deposition of extracellular type II collagen fibrils. Theextracellular localization of the monoclonal antibody III-D5 against CMPwas compared with that of a polyclonal antiserum to type II collagen. Adense filamentous extracellular matrix pattern was observed for both CMPand type II collagen. In a separate experiment the extracellularlocalization of CMP was contrasted with that of aggrecan. Thefilamentous distribution of extracellular CMP did not co-localize withthe amorphous pattern of aggrecan. Immunofluorescent staining ofchondrocyte cultures carrying the genetic defect nanomelia, which ischaracterized by the absence of aggrecan in the cartilage extracellularmatrix, demonstrated a similar co-localization of CMP with type IIcollagen.

There was no apparent co-localization of CMP and aggrecan in cultures ofnormal chondrocytes. However, material remaining after treatment withtesticular hyaluronidase reacted with antibodies to the aggrecan coreprotein and co-localized with the filamentous CMP/type II collagenpattern.

To examine the ultrastructural associations of CMP with the cartilageextracellular matrix, chondrocytes in culture were examined afterimmunoperoxidase reactions. A periodic distribution of CMP along thenetwork of collagen fibrils was observed at both low and highmagnification. The ultrastructural immunostaining pattern for type IIcollagen was similarly periodic. Quantitative analysis revealed aperiodic repeat of 59.3 nm (standard error=±0.6 nm) for CMP and 60.3 nm(standard error=±0.6 nm) for type II collagen. This observed repeat isconsistent with the 60-65 nm periodicity known to be characteristic oftype II collagen. The normal serum control appeared non-reactive.

Cell Culture

Chondrocytes were obtained from the sterna of 15 day-old White Leghornchicken embryos as described by Cahn et al., Methods in DevelopmentBiology, Wilt et al. (Eds.), Thomas Y. Crowell Co., New York pp. 493-530(1967). For immunofluorescence studies, cells were cultured in monolayeron gelatinized, carbon coated-coverslips at a density of 6×10⁵ cells per60 mm tissue culture dish in 3 ml Ham's F-12 medium containing 10% fetalbovine serum and 1% antibiotic-antimycotic mix. Cultures were fed freshmedium containing 50 mM ascorbic acid on days 3 and 4 and fed again 2 hrbefore fixation in 75% ethanol. For immunoelectron microscopy, cellswere cultured on gelatinized 35 mm tissue culture dishes at equivalentdensities in 1.5 ml of the same medium. Cell cultures were fed freshmedium with ascorbate as described above prior to fixation with 2.5%glutaraldehyde on day 5.

Immunofluorescent Staining

At 5 days of culture, chondrocytes were washed several times with Hank'sBalanced Salt Solution (HESS), fixed with 75% ethanol and prepared forimmunofluorescence staining as described in Vertel et al., J. Cell.Biochem. 27:215 (1985). In some experiments, glycosaminoglycans wereremoved from extracellular aggrecan prior to fixation by a briefdigestion with testicular hyaluronidase (Vertel et al., 1985).

Cells were incubated for 20 min. at room temperature with primaryantibodies and FITC or TRITC-coupled secondary antibodies as indicatedin the figure legends. Cells were washed extensively with HBSS betweenantibody incubations. After further washes with HBSS, the coverslipswere mounted in phosphate buffer/glycerol (1:9, v/v). Samples wereobserved and photographed using a Leitz Ortholux microscope with phaseand epifluorescence optics. Fields were selected from double-stainedspecimens and photographed sequentially for FITC and TRITC staining.

Immunoelectron Microscopy

After 5 days of culture as described above, chondrocytes were fixed in2.5% glutaraldehyde for 30 min. Immunoperoxidase studies with mousemonoclonal III-D5 anti-CMP and rabbit anti-type II collagen wereconducted according to the procedure of Brown et al., (Cell 36:295,1984), with some modifications (Vertel et al., J. Cell. Biol. 108:1327,1989). Samples were counterstained for 5 min with Reynolds lead citrateand observed and photographed using a Zeiss 10 electron microscope.

Analysis of the periodicity of CMP and type II collagen immunostainingwas performed using a computer assisted image analysis system andMicrocomp software. Actual readings were taken directly fromphotographic prints of electron micrograph negatives magnified 4.65x.Seven samples were measured from each enlargement. Measurements of over200 samples were used in each group. Absolute calibrations were based onthe use of a carbon replica standard. All electron micrographs andenlargements were photographed, developed, and printed at the samemagnifications during the same photographic sessions.

Electrophoresis

SDS-PAGE (Laemmli, 1970) was carried out on gels of various acrylamideconcentrations, as indicated in the figure legends, blotted (Towbin etal., 1979) to Immobilon-P (Millipore Corp., Bedford, Mass.), and stainedusing the above antibodies followed by alkaline phosphatase conjugatedgoat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) (BioradLaboratories) as appropriate, and using6-bromo-4-chloro-3-indolyphosphate p-toluidine salt/nitrobluetetrazolium chloride (Gibco BRL, Gaithersburg, Md.) as chromogenicsubstrate.

CMP Influences Fibrillogenesis

A fibrillogenesis assay (performed as described below) demonstrated thatCMP affects fibrillogenesis. Referring to FIG. 1, which depicts theresults of an assay in which fibrillogenesis is monitored by measuringturbidity of a type II collagen solution in the presence and absence ofCMP, CMP substantially increased turbidity. Similar results wereobserved using type I collagen. In both instances microscopicexamination revealed that fibrils formed in the presence of CMP werethinner than those formed in the absence of CMP.

Fibrillogenesis Assay

The assay was performed essentially as described by Hedbom et al. (J.Biol. Chem. 264:6898, 1989). Briefly, fibrillogenesis was initiated bythe addition of type II collagen to fibrillogenesis buffer (60 mM NaCl,30 mM NaPO₄ pH 7.3!) in the presence or absence of CMP. The final typeII collagen concentration was 200 μg/ml, the final CMP concentration was20 μg/ml. Fibrillogenesis was monitored by periodically measuring theoptical density of the mixture at 400 nM over the course of severalhours. The reaction was carried out at 37° C.

CMP-1 and CMP-2 Mediate Type II Collagen Binding

CMP-1 (amino acids 30 to 220 of CMP) and CMP-2 (amino acids 262-450 ofCMP) are important for collagen binding. An in vitro assay, performed asdescribed above, demonstrated that both CMP-1 and CMP-2 can bind type IIcollagen. In these experiments various portions of CMP coding sequencewere used to generate E. coli maltose binding protein (MBP)/CMP fusionproteins (New England Biolabs, Beverly, Mass.). The fusion proteins wereexpressed in E. coli, and purified using an amylose-sepharose column.Eluate was applied to plates of immobilized type II collagen. Fusionproteins were then detected using either anti-maltose binding protein oranti-CMP monoclonal antibodies in combination with an alkalinephosphatase conjugated secondary antibody.

FIG. 2 illustrates the results of assays of three different fusionproteins. The relative amount of binding is shown as a function of theconcentration of fusion protein (in μg/ml) for MBP fused to amino acids1-450 of chicken CMP (first bar in each group); MBP fused to chickenCMP-1 (second bar in each group); and MBP fused to amino chicken CMP-2(third bar in each group). Binding was detected with anti-MBP antiserum.

Collagen Binding Assay

Plates were coated with a 1:75 dilution in PBS-T of 2 mg/ml type IIcollagen (Nitta Gelatin Inc., Osaka, Japan) and then dried for ca. 18 hrat 37° C. Fusions proteins, purified as described above, were applied tothe collagen coated plates and incubated for 1 hr at 37° C. Fusionproteins were detected with anti-MBP antiserum (New England Biolabs,Beverly, Mass.) or anti-CMP antibody (described above) and a secondaryantibody essentially as described above.

Collagen-Binding Fragments of CMP-1 and CMP-2

Construction of MBP-CMP-1 and MBP-CMP-2 Fusion Proteins.

The CMP-1 domain was synthesized by PCR using a 5' primer sequencecorresponding to amino acid residue 24 (first amino acid of the matureprotein) to amino acid residue 30 and a 3' primer sequence correspondingto amino acid residues 214 through 220. The CMP-2 fragment was amplifiedby using a 5' primer sequence corresponding to amino acid residues262-268, and a 3' primer sequence corresponding to amino acid residues444-450. The PCR fragments were cloned by blunt end ligation into theStuI cloning site of the maltose binding protein encoding vector pMAL-C2(New England Biolabs, Beverly, Mass.). Miniprep DNA samples weredigested with the restriction enzyme BamHI (BamHI sites are located inthe MBP polylinker at the 5' and 3' ends of the inserted CMP fragment)and the appropriate clones were screened for the presence of insertfragments of the expected size. Positive clones were next screened forexpression of fusion protein by Western blotting of SDS-PAGE separatedproteins and testing for cross reactivity with anti-CMP antibodies andalkaline phosphatase linked secondary antibodies.

Expression and purification of MBP-CMP-1 and MBP-CMP-2 Fusion Proteins

The expression of CMP-1 or CMP-2 fusion proteins in MBP vectors wasinduced by addition of IPTG to cultures of logarithmically growing E.coli cultures as described in the New England Biolabs pMAL protocol. Theexpressed protein was purified on amylose resin as described in the NewEngland Biolabs protocol. Briefly, the cells were pelleted bycentrifugation (5000 RPM, in Sorvoll SS 34 rotor, 10 Min. at 4° C.),washed in column buffer (20 mM Tris-Cl pH 7.4, 200 mM NaCl, mM EDTA, 1mM EGTA, 1 mM DTT, 1 mM sodium azide), lysed in the same buffer byresuspending cells in 1/100th of the original culture volume andsonication. The cell debris was pelleted by centrifugation at 20,000 gfor 30 minutes. The supernate was applied to an amylose column using 50ml of resin for each one liter of bacterial culture. The column waswashed with 10 column volumes of column buffer, and the bound fusionprotein was eluted with the column buffer supplemented with 10 mMmaltose.

Construction of Deletions of MBP-CMP1 and MBP-CMP2

Deletions of MBP-CMP1 and MBP-CMP2 were made in order to allow moreprecise delineation of the collagen binding sites of CMP. The MBP-CMP2(or MBP-CMP1) plasmid was linearized at its unique XbaI site (this siteis located in the polylinker sequence of pMLA-c at the 3' of the CMP2(or CMP1) fragment). The linearized plasmid was digested with Bal31exonuclease (at an enzyme concentration that theoretically would digestDNA at the rate 100 base pairs per minute per end) and aliquots of thereaction mixture removed at one minute intervals. The Bal31 digestionwas stopped by addition of EGTA. Phenol and phenol/chloroformextractions were performed and the DNA ethanol precipitated. The ends ofthe DNA were repaired by Klenow, and ligated with 50 molar excess of thePharmacia Suppressible Termination Linker (Pharmacia, Piscataway, N.J.).The sequence of this oligonucleotide contains a translation stop codonin each of the three reading frames as well as introducing a unique XbaIrestriction site into the clones. E. coli XL1 Blue cells (Stratagene,San Diego, Calif.) were transformed with the ligated DNA byelectroporation. The colonies were screened for presence of shortenedMBP-CMP2 plasmids by digestion of miniprep DNA with XbaI. Putativeclones containing shortened plasmids were induced by IPTG and screenedfor expression of fusion proteins by SDS-PAGE. Fusion proteins werepurified from selected colonies and screened (by ELISA) for the abilityto bind to type II collagen.

Detection of Collagen Binding by CMP Fragments

The measurement of the binding of the CMP constructs to collagen wereperformed using the ELISA methods described above.

Determination of the Molecular Weights of MBP-CMP and MBP-CMP andDeletion Fusion Proteins

The molecular weight of the purified MBP-CMP-1 and MBP-CMP-2 fusionproteins and their Bal31-generated truncated versions were estimatedfrom the distance migrated by the proteins after electrophoresis on 10%polyacrylamide gels. Protein standards with known molecular weights wereelectrophoresed in separate lanes of the same gel. The molecular weightsof the standard were plotted as a function of their distance migrated.The molecular weights of the MBP fusion proteins were estimated from thedistance migrated on these gels.

Determination of molecular weight of and the number of amino acids inthe CMP regions of the MBP-CMP fusion proteins

Since the fusion proteins include both the MBP (43,000 daltons) and aCMP domain (or fragment thereof), the molecular weight of the CMPcomponent of a fusion protein was determined by subtracting themolecular weight of the MBP from that of the fusion protein. This netmolecular weight (i.e. that of the CMP component) was divided by theaverage molecular weight of an amino acid (110) to obtain an estimate ofthe number of amino acids of the CMP domains.

Identification of a region in CMP-1 and CMP-2 which is responsible forbinding to collagen

Fusion genes encoding MBP-CMP-1 and MBP-CMP-2 were subjected to Bal31digestion to generate terminal deletions in the CMP-domain-encodingregion of the fusion gene. Fusion proteins, with truncated CMP domains,were tested for the ability to bind collagen. As is shown in Table I,deletion of up to 145 residues from the C-terminal end of the CMP domainof the fusion protein did not prevent binding to collagen. Thus,approximately 45 amino acid residues at the N-terminal end of CMP-1 andCMP-2 is or contains a region critical to collagen binding. Thisapproximately 45 amino acid residue region corresponds approximately toamino acid residues 24-68 of CMP-1 (A P P Q P R G T L C R T K P T D L VF I I D S S R S V R P Q E F E K V K V F L S R V I E G (SEQ. ID. NO: 38))and to amino acid residues 262-306 of CMP-2 (A C S G G S G S A L D L V FL I D G S K S V R P E N F E L V K K F I N Q I V E S L E V S E (SEQ. ID.NO: 39)) in the chicken protein (by the numbering system of Kiss et al.)and to amino acid residues 27-71 of CMP-1 (A P Q S R G H L C R T R P T DL V F V V D S S R S V R P V E F E K V K V F L S Q V I E S L D (SEQ. ID.NO: 40)) and to amino acid residues 264-309 of CMP-2 (V C S G G G G S SA T D L V F L I D G S K S V R P E N X E L V K K F I S Q I V D T L D V SD (SEQ. ID. NO: 41)) in the human protein (by the numbering system ofJenkins et al.).

The collagen binding sequence 1 (CBS1) motif

Within the 45 amino acid region is a smaller region of approximately 8to 10 residues in length, referred to as The collagen binding sequence 1(CBS1) motif, which is common to many proteins which bind collagen. Itssequence, in the chicken CMP-1 gene is T-D-L-V-F-I-I-D-S-S (SEQ. ID. NO:42). Its sequence, in the chicken CMP-2 gene is L-D-L-V-F-L-I-D-G-S(SEQ. ID. NO: 43). The sequence is located at approximately amino acidresidues 38-47 of CMP-1 and amino acid residues 271-280 of CMP-2 in thechicken protein (by the numbering system of Kiss et al.) and atapproximately amino acid residues 40-49 of CMP-1 and amino acid residues274-283 of CMP-2 in the human protein (by the numbering system ofJenkins et al.).

                                      TABLE I                                     __________________________________________________________________________                     Molecular Wt                                                                  of CMP Fragment                                                       Estimated                                                                              Est. m.w. of                                                                         Estimated                                                     Molecular Wt.                                                                         fusion protein-                                                                       Number of                                            Fusion Protein                                                                         Of Fusion                                                                             m.w. of MBP                                                                           Amino Acids                                                                           Collagen                                     Clone Number                                                                           Protein (43,000)!                                                                             in CMP Fragment                                                                       Binding                                      __________________________________________________________________________    MBP-CMP1 67,000  24,000  218     YES                                          MBP-CMP1-Δ22.1                                                                   48,000   5,000  45      YES                                          MBP-CMP2 68,000  25,000  227     YES                                          MBP-CMP2-Δ4.1                                                                    58,000  15,000  136     YES                                          MBP-CMP2-Δ40.1                                                                   53,000  10,000  91      YES                                          MBP-CMP2-Δ23.2                                                                   48,000   5,000  45      YES                                          MBP-CMP2-Δ46.2                                                                   43,000     0    0       NO                                           MBP-CMP2-Δ47.2                                                                   43,000     0    0       NO                                           MBP      43,000     0    0       NO                                           __________________________________________________________________________

USE

The collagen-binding CMP polypeptides of the invention may be used todeliver therapeutic, diagnostic, cosmetic or other compounds tocollagenous tissue. For example an imaging agent such as a fluorescentor radioactive label can be attached to a collagen-binding CMPpolypeptide and the conjugate can be used to visualize and analyze thestructure of collagenous tissue. A therapeutic agent capable ofinfluencing the structure of collagenous tissue, e.g., collagenase, maybe attached to collagen-binding CMP polypeptide and thus delivered tocollagenous tissue. In a similar manner an anti-cancer drug may bedelivered to collagenous tissue. Compounds designed for cosmeticpurposes, e.g, for increasing hydration, may be delivered to collagenoustissue, e.g., skin, by covalent attachment to a collagen-binding CMPpolypeptide.

To effect delivery a compound, e.g., a protein, is generally covalentlyattached to a collagen-binding CMP peptide. Many techniques forcovalently linking polypeptides are known to those skilled in the art.For example,succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene andN-succinimidyl 3-(2-pyridyldthio)propionate (Pierce, Rockford, Ill.) areheterobifunctional cross-linkers which can be used to link proteins in astep-wise fashion avoiding the formation of homopolymers and otherundesirable side reactions. Alternatively genetically-engineered fusionproteins can be created to link CMP and a protein to be delivered tocollagenous tissue. In addition the amino acid sequence of CMP, orpolypeptide fragments thereof may be modified to permit attachment ofparticular compounds. For example, site-directed mutagenesis could beused to introduce an attachment site for glycosaminoglycan in CMP. TheEGF-like domain could modified to introduce such an attachment site.Modification of the EGF-like domain will not be apt to interfere withbinding of CMP to collagen.

Polypeptides of the invention can be applied to a surface to promote theattachment (e.g., by covalent or noncovalent association) of collagen tothe surface, e.g., the surface of a tissue, e.g., the surface of a toothor gum, or the surface of a medical device, e.g., a device to beimplanted in the body.

CMP or fragments thereof which affect fibrillogenesis can be used toform collagen fibrils. Fibrils so formed can be used as a prosthetic toreplace components of skin or other collagenous tissue or as a coatingmaterial that will enhance biocompatibility of prosthetic devices.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 43                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ThrAspLeuValPheIleIleAspSerSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuAspLeuValPheLeuIleAspGlySer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ThrAspLeuValPheValValAspSerSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ThrAspLeuValPheLeuIleAspGlySer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspIleValPheLeuThrAspAlaSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspIleValLeuLeuValAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AspLeuValPheLeuValAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AspValValPheLeuValAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AspLeuValPheLeuValAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AspLeuValPheIleIleAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AspLeuValPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AspIleMetLeuLeuValAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AspLeuLeuPheValLeuAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AspLeuPhePheValLeuAspThrSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AspIleValPheLeuLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AspIleMetPheValIleAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AspIleAlaPheIleMetAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GluLeuAlaPheAlaIleAspThrSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AspValIleLeuGlyPheAspValSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AspIleValPheLeuLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AspValValPheLeuIleAspSerSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AspValValPheLeuValAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AspValValPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AspIleLeuPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AspIleIlePheLeuLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AspIleValPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AspLeuIlePheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AspLeuValPheLeuLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      AspValAlaPheValLeuGluGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AspValIleLeuLeuLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AspIleValPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AspIleValPheLeuIleAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AspLeuValPheLeuPheAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      AspIleValIleValLeuAspGlySer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AspValValLeuValCysAspGluSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AspIleSerPheLeuLeuAspValSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 496 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      MetArgValLeuSerGlyThrSerLeuMetLeuCysSerLeuLeuLeu                              151015                                                                        LeuLeuGlnAlaLeuCysSerProGlyLeuAlaProGlnSerArgGly                              202530                                                                        HisLeuCysArgThrArgProThrAspLeuValPheValValAspSer                              354045                                                                        SerArgSerValArgProValGluPheGluLysValLysValPheLeu                              505560                                                                        SerGlnValIleGluSerLeuAspValGlyProAsnAlaThrArgVal                              65707580                                                                      GlyMetValAsnTyrAlaSerThrValLysGlnGluPheSerLeuArg                              859095                                                                        AlaHisValSerLysAlaAlaLeuLeuGlnAlaValArgArgIleGln                              100105110                                                                     ProLeuSerThrGlyThrMetThrGlyLeuAlaIleGlnPheAlaIle                              115120125                                                                     ThrLysAlaPheGlyAspAlaGluGlyGlyArgSerArgSerProAsp                              130135140                                                                     IleSerLysValValIleValValThrAspGlyArgProGlnAspSer                              145150155160                                                                  ValGlnAspValSerAlaArgAlaArgAlaSerGlyValGluLeuPhe                              165170175                                                                     AlaIleGlyValGlySerValAspLysAlaThrLeuArgGlnIleAla                              180185190                                                                     SerGluProGlnAspGluHisValAspTyrValGluSerTyrSerVal                              195200205                                                                     IleGluLysLeuSerArgLysPheGlnGluAlaPheCysValValSer                              210215220                                                                     AspLeuCysAlaThrGlyAspHisAspCysGluGlnValCysIleSer                              225230235240                                                                  SerProGlySerTyrThrCysAlaCysHisGluGlyPheThrLeuAsn                              245250255                                                                     SerAspGlyLysThrCysAsnValCysSerGlyGlyGlyGlySerSer                              260265270                                                                     AlaThrAspLeuValPheLeuIleAspGlySerLysSerValArgPro                              275280285                                                                     GluAsnPheGluLeuValLysLysPheIleSerGlnIleValAspThr                              290295300                                                                     LeuAspValSerAspLysLeuAlaGlnValGlyLeuValGlnTyrSer                              305310315320                                                                  SerSerValArgGlnGluPheProLeuGlyArgPheHisThrLysLys                              325330335                                                                     AspIleLysAlaAlaValArgAsnMetSerTyrMetGluLysGlyThr                              340345350                                                                     MetThrGlyAlaAlaLeuLysTyrLeuIleAspAsnSerPheThrVal                              355360365                                                                     SerSerGlyAlaArgProGlyAlaGlnLysValGlyIleValPheThr                              370375380                                                                     AspGlyArgSerGlnAspTyrIleAsnAspAlaAlaLysLysAlaLys                              385390395400                                                                  AspLeuGlyPheLysMetPheAlaValGlyValGlyAsnAlaValGlu                              405410415                                                                     AspGluLeuArgGluIleAlaSerGluProValAlaGluHisTyrPhe                              420425430                                                                     TyrThrAlaAspPheLysThrIleAsnGlnIleGlyLysLysLeuGln                              435440445                                                                     LysLysIleCysValGluGluAspProCysAlaCysGluSerLeuVal                              450455460                                                                     LysPheGlnAlaLysValGluGlyLeuLeuGlnAlaLeuThrArgLys                              465470475480                                                                  LeuGluAlaValSerLysArgLeuAlaIleLeuGluAsnThrValVal                              485490495                                                                     (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AlaProProGlnProArgGlyThrLeuCysArgThrLysProThrAsp                              151015                                                                        LeuValPheIleIleAspSerSerArgSerValArgProGlnGluPhe                              202530                                                                        GluLysValLysValPheLeuSerArgValIleGluGly                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      AlaCysSerGlyGlySerGlySerAlaLeuAspLeuValPheLeuIle                              151015                                                                        AspGlySerLysSerValArgProGluAsnPheGluLeuValLysLys                              202530                                                                        PheIleAsnGlnIleValGluSerLeuGluValSerGlu                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AlaProGlnSerArgGlyHisLeuCysArgThrArgProThrAspLeu                              151015                                                                        ValPheValValAspSerSerArgSerValArgProValGluPheGlu                              202530                                                                        LysValLysValPheLeuSerGlnValIleGluSerLeuAsp                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      ValCysSerGlyGlyGlyGlySerSerAlaThrAspLeuValPheLeu                              151015                                                                        IleAspGlySerLysSerValArgProGluAsnXaaGluLeuValLys                              202530                                                                        LysPheIleSerGlnIleValAspThrLeuAspValSerAsp                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ThrAspLeuValPheIleIleAspSerSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      LeuAspLeuValPheLeuIleAspGlySer                                                1510                                                                          __________________________________________________________________________

Other embodiments are within the following claims.

What is claimed is:
 1. A chimeric polypeptide which includes a fragmentof cartilage matrix protein domain CMP-1 or CMP-2, said fragmentincluding a collagen binding site and said fragment being at least 99%homologous with the corresponding residues of cartilage matrix proteindomain CMP-1 or CMP2.
 2. The chimeric polypeptide of claim 1, whereinsaid fragment is 100% homologous with the corresponding residues ofcartilage matrix protein domain CMP-1 or CMP2.
 3. The chimericpolypeptide of claim 1, wherein said fragment is 100% homologous withthe corresponding residues of cartilage matrix protein domain CMP-1 orCMP2.
 4. A molecular conjugate comprising the chimeric polypeptide ofclaim 1 and an imaging or therapeutic agent.